Simultaneous Determination of Mono‐, Di‐, and Trihydroxyoctadecenoic Acids in Beer and Wort

Monohydroxy, dihydroxy‐, and trihydroxyoctadecenoic acids in beer and wort were simultaneously determined using gas chromatography after a solid extraction method. These three acids were detected at ppm levels in the wort. The monohydroxyoctadecenoic acids were not detected after wort boiling, but the dihydroxy‐ and trihydroxyoctadecenoic acids were transferred through wort boiling, fermentation and lagering into the finished beer. During the mashing using a laboratory mash bath, they gradually increased to about twice the levels those at mashing‐in. The amounts of dihydroxyoctadecenoic acid and trihydroxyoctadecenoic acid in commercial beer samples varied from 0.6 to 1.6 ppm and 6 to 15 ppm, respectively.     

MASHING EQUIPMENT SIMULATING DEEP-BED PRODUCTION MASHES

A glass mashing unit, using only 1 kg of grist, has been designed to simulate the effects of wort run-off from a deep infusion tun. The apparatus incorporates automatic sparging control and measurement of mash temperature without disturbing the filtration characteristics of the mash bed. The ease of wort run-off is assessed by monitoring the pressure differential across the mash bed while wort is allowed to separate at a pre-determined flow rate. The depth of mash bed (1·8 metres) is equivalent to that in a commercial infusion tun and, although only a small quantity of grist is required, the apparatus permits characterisation of the potential run-off behaviour of different malts under production conditions.     

LIPIDS IN WORT

Material balances have been constructed for lipids during wort preparation in commercial breweries. 4–5% of malt lipids were released into the wort using the mash filter, 1·0% with the lauter tun but only 0–3% with the mash tun. Up to 30% of malt lipids may be oxidised during mashing. Hop products make a contribution to the lipids in the copper though these lipids are not readily extracted. Efficient removal of spent hops and hot break will ensure a low level of wort lipids in the fermentation vessel.     

The Use of an Extracellular Ferulic Acid Esterase from Lactobacillus acidophilus K1 for the Release of Phenolic Acids During Mashing

A purified extracellular ferulic acid esterase from Lactobacillus acidophilus K1 was successfully used during mashing for the release of free phenolic acids into sweet wort. The enzyme was produced in bioreactors and partially purified to obtain the monoenzyme preparation. Release of free ferulic and vanillic acid into the wort at 52°C (with the use of 4.09–14.60 enzyme activity units/L of the mash) and ferulic acid at 62°C (14.60 units/L) was observed. Free p‐OH‐benzoic and syringic acids were effectively released at 26°C at each enzyme concentration used. Free p‐OH‐benzoic acid was also released by the enzyme (14.60 U/L) at 52°C‐74°C. Free protocatechuic acid was effectively hydrolyzed by the enzyme preparation (8.75 U/L and 14.60 U/L) at 26°C‐52°C. Free caffeic acid (effectively released at 26°C‐62°C) originated from chlorogenic acid. No p‐coumaric acid was released due to the action of bacterial esterase during mashing. Ferulic acid esterase from L. acidophilus K1 exhibited no ability to release free phenolic acids during mashing at 62°C or at 74°C due to its low thermostability. In conclusion, L. acidophilus K1 is an attractive source for the production of ferulic acid esterase, which can be useful for the release of antioxidant phenolic acids in the early stages of mashing.     

INVESTIGATION OF TRANSAMINASE ACTIVITY DURING MASHING

Malt peptidase activity has been determined by Jones & Pierce as the difference in quantities of free amino acids produced under standard conditions between an enzymic mash and one in which the enzymic activity has been destroyed with boiling absolute alcohol. The measurement is a reasonable assessment of individual mash tun peptidase activity provided no interconversion of amino acids takes place during the mashing. Any such interconversion would involve the participation of transmination reactions in the malt. As a means of detecting transaminase activity, malt was mashed at 50° C. in the presence of ¹⁵N-l -glutamic acid and ¹⁵N-l -alanine, and the extracted amino acids were analysed for ¹⁵N by mass spectrometry. Little transaminase activity was detected.     

SMALL SCALE MASHING EXPERIMENTS WITH GRISTS CONTAINING HIGH PROPORTIONS OF RAW SORGHUM

Small scale mashes (50 g total grist) with grists containing high proportions of raw sorghum (50%–80% malt replacement) showed high values of extract recovery and produced worts of lower total nitrogen, free amino nitrogen, viscosity and colour but higher values of pH compared to worts produced from all malt mashes. Increasing the proportion of raw sorghum in the grist relative to malt resulted in a decline in extract recovery, wort total nitrogen, free amino nitrogen and an increase in wort pH. Addition of industrial enzyme preparations to mashes containing raw sorghum resulted in higher values of extract recovery (enzyme preparations containing α amylase and β glucanase), higher values of wort total nitrogen and free amino nitrogen (enzyme preparations containing a neutral proteinase) and decreased wort viscosity (enzyme preparations containing β glucanase or cellulases) compared to worts produced from untreated mashes. Worts and beers were produced on a pilot brewery scale from 50% malt and 50% polished (whole) sorghum (single decoction mashing regime) and 20% malt and 80% raw sorghum supplemented with an industrial enzyme preparation (double mashing regime). Mashes comprising 50% malt and 50% polished sorghum showed comparable wort filtration behaviour (lautering) to that of control mashes (70% malt and 30% maize grists) whereas wort produced from 20% malt and 80% raw sorghum filtered slowly. Worts produced from grists containing sorghum were of high fermentability and showed lower levels of total nitrogen and free amino nitrogen compared to control worts. Analysis of worts produced from small scale mashes containing raw sorghum and a pilot brewery scale mash comprising 20% malt and 80% raw sorghum demonstrated that the levels of total nitrogen and free amino nitrogen were higher than expected from the reduction in the malt content of the mash, consistent with the release of nitrogenous components (polypeptides, peptides and amino acids) derived from sorghum into the wort. Beers produced from 50% malt and 50% polished sorghum and 20% malt and 80% raw sorghum were filtered without difficulty and were of sound flavour. Beers produced from 50% malt and 50% polished sorghum contained lower levels of isobutanol, 2-methylbutanol, dimethylsulphide and higher levels of n propanol and diacetyl compared to control beers.     

DETERMINATION OF FATTY ACID HYDROPEROXIDES PRODUCED DURING THE PRODUCTION OF WORT

Linoleic and linolenic acid hydroperoxides in malt, mash, or wort were determined with high sensitivity and high selectivity by the chemiluminescence-high performance liquid chromatography (CL-HPLC) method using isoluminol-microperoxidase solution as a luminescing reagent. The determination limit of this method for both hydroperoxides was 0.1 μM in mash or wort. During the mashing in a laboratory mash bath, the hydroperoxides started to increase just after mashing-in, reached a maximum at 65°C, and then decreased. Though the hydroperoxides were detected in mash just before the lautering in a pilot scale brewing, they disappeared during the lautering and could not be detected during the subsequent stages of wort production. Therefore, it was thought that the mashing process is the most important of the lipid oxidation reactions during wort production. It is also expected that the CL-HPLC method can give useful information on lipid oxidation mechanisms during wort production.     

Dry and Wet Milling of Malt. A Preliminary Study Comparing Fermentable Sugar,Total Protein,Total Phenolics and the Ferulic Acid Content in Non‐Hopped Worts

In this work, fermentable sugar, total protein, phenolics and ferulic acid content were estimated in sweet worts at different points of lautering. Transfer of these selected malt compounds into worts was analyzed in relation to the method of malt milling (wet milling of malt — the “test worts” or dry milling of malt — the “reference worts”). Glucose, maltose and maltotriose were more rapidly transferred into sweet worts at the early stages of lautering (40 hL and/or 80 hL of wort) after wet milling in comparison to dry milling. Total protein content in the test worts was significantly higher than in the corresponding reference worts at each stage of lautering. Transfer of phenolic compounds and ferulic acid (in the free as well as in the ester form) from the mash into sweet worts was significantly improved by dry milling, but not by wet milling. No difference in the total antioxidant activity was observed between the two types of worts. In conclusion, it can be stated that wet conditioning of malt before milling enhances the fast transfer of fermentable sugars and proteins from the mash into the sweet wort during lautering. Lautering is a time‐consuming process, and time reduction without the loss of wort quality should be a priority. Therefore, wet milling can be of interest to professionals in the field as an interesting alternative method to improve the mashing process.     

Impact of full-scale brewing processes on lager beer nitrogen compounds

This paper analyses in a full-scale industrial process firstly the nitrogen compounds in all-malt and in maize-adjunct worts, then their fate during the main brewing steps and finally the influence on them of two different separation technologies: mash filter (Meura 2001) and lauter tun (Steinecker FVAS 26). Data showed that (1) maize-adjunct worts have a lower total nitrogen compounds than all-malt worts; (2) assimilable nitrogen represents 20–24% out of the total nitrogen in both all-malt and in adjunct worts; (3) free amino nitrogen nearly doubles in all-malt compared with adjunct worts; (4) proline and asparagine are the most abundant amino acids in both worts; (5) ammonium disappears during fermentation in wort with the lowest nitrogen content, i.e. in maize-adjunct wort. Moreover, the total nitrogen is reduced in all-malt by 80% with the Steinecker FVAS 26 lauter tun and 25% with the Meura 2001 filter, while in adjunct worts by 87% with the Steinecker FVAS 26 lauter tun and 29% with the Meura 2001 filter. After mash filtration, the content of assimilable nitrogen remains to be adequate for an efficient fermentation, but after lauter tun separation, the assimilable nitrogen reaches values that may compromise the regular fermentation process in both all-malt and adjunct worts. Therefore, when using lauter tun, we have to intervene to reduce its impact on nitrogen compounds and/or plan the wort nitrogen supplementation to overcome the stuck and sluggish fermentations.     

RATE-LIMITING ENZYMES IN THE LIBERATION OF AMINO ACIDS IN MASHING

When 29 malts representing a wide variation in free amino nitrogen of wort were mashed for 3 h at 45°C, the increases in TCA-soluble amino nitrogen varied from 47 to 116 μmoles per gramme of malt. The carboxypeptidase activities of the malts showed only small positive correlations with the increase of amino nitrogen during mashing. Inactivation of the different malt carboxypeptidases by 70 to 90% with diisopropylfluorophosphate decreased the liberation of amino nitrogen by only 5 to 25%. Moreover, addition of purified malt carboxypeptidase I to the mashing suspensions had no effect on the liberation of amino nitrogen. It is therefore evident that the carboxypeptidases do not have a major rate-limiting role in the liberation of amino acids in mashing, although it has earlier been shown that they are essential enzymes in this process. The proteinase activities of the malts, determined using two methods, correlated strongly with the liberation of amino nitrogen in mashing. Addition of crude malt proteinase to the mash increased, and addition of purified barley proteinase inhibitor decreased the measured proteinase activity and the liberation of amino acids roughly to the same extent. These results indicate that the most important rate-limiting enzyme activity in the liberation of amino acids in mashing is the proteinase activity of the malt.     

Behavior of mono-, di-, and trihydroxyoctadecenoic acids during mashing and methods of controlling their production

The behavior of mono-, di-, and trihydroxyoctadecenoic acids was investigated during laboratory-scale mashing under various conditions with a view to controlling their production. Using a malt in which the lipoxygenase activity was at only a trace level (less than 0.01 U/g) or starting the mashing at a higher temperature than that conventionally used (65 degrees C instead of 48 degrees C) significantly decreased the production of these hydroxy fatty acids. Lowering the pH of the mash to inhibit lipoxygenase activity and preventing O2 uptake by the mash using carbon dioxide were also effective in reducing the amounts of these acids produced during mashing. From the viewpoint of industrial-scale beer production, the prevention of O2 uptake by the mash was selected as an appropriate method for reducing oxidation during wort production without affecting the subsequent brewing process or the taste of the finished beer. After introducing oxidation prevention procedures, the content of trihydroxyoctadecenoic acids decreased by about 30% and the foam stability and taste were improved in commercial products brewed using less than 25% malts.     

Vitamins in brewing: the impact of wort production on the thiamine and riboflavin vitamer content of boiled sweet wort

Wort production contains a number of processing steps that are aimed at the optimal extraction of nutrients from malt, including vitamins. This research revealed that the different wort production processing steps imposed different influences on the thiamine and riboflavin vitamer content of the final sweet wort. These vitamins play vital roles within yeast metabolism, where they act as enzyme cofactors. As such thiamine vitamers play a crucial role in many decarboxylating enzymes, while riboflavin vitamers play an integral role in energy production and redox maintenance. While mashing releases valuable starch into the liquor, both thiamine and riboflavin are also extracted. The extraction of these vitamins is the greatest at 65°C and is indirectly linked to the amylase activity. When the starches are broken down during mashing, the thiamine and riboflavin vitamers are gradually released into the mash liquor. The boiling and trub removal (whirlpool) processes impose losses in both vitamins owing to the high temperatures exhibited during these stages. While hop pellets were shown to contribute a small proportion of the vitamers studied, the use of kettle finings caused a significant reduction in both thiamine and riboflavin vitamers. Copyright © 2014 The Institute of Brewing & Distilling     

HYDROPHOBIC POLYPEPTIDE EXTRACTION DURING HIGH GRAVITY MASHING— EXPERIMENTAL APPROACHES FOR ITS IMPROVEMENT

The aim was to establish if a substantial increase in hydrophobic polypeptides could be achieved during high gravity mashing. When worts with gravities ranging from 5–20°P were analysed for hydrophobic polypeptide content it was found that there was no appreciable increase in hydrophobic polypeptide levels. Remashing of the spent grains from low and high gravity mashes demonstrated that this resulted from inefficient extraction of hydrophobic polypeptide levels during the mashing process. For example, wort produced from remashed high gravity spent grains contained 150 mg/L hydrophobic polypeptides compared to only 10 mg/L in the low gravity remashed spent grains. Experiments were conducted, employing standard mashing techniques, in an attempt to increase the extraction of hydrophobic polypeptides during high gravity mashing. Thus the use of gypsum, proteolytic stands, varying liquor to grist ratios and wheat malt addition were all investigated for their effect on hydrophobic polypeptide extraction during high and low gravity mashing. Wort analysis demonstrated that none of the techniques employed had a significant effect on hydrophobic polypeptide extraction. When wort from remashed spent grains was used as mashing in liquor for a fresh mash and the resultant worts analysed for hydrophobic polypeptides it was observed that no increase in hydrophobic polypeptide extraction was achieved. For example, wort from the remashed high gravity spent grains, containing 140 mg/L hydrophobic polypeptides, when used as mashing-in liquor, produced no increase in hydrophobic polypeptide levels in the resultant high gravity wort (230 mg/L) when compared to a high gravity wort produced using distilled water as mashing-in liquor (255 mg/L). It is therefore concluded that a saturation point has been reached and no more hydrophobic polypeptides can be extracted during mashing regardless of the procedures employed.     

外加酶法酿制低糖啤酒糖化工艺的研究

以普鲁兰酶Ｐｒｏｍｏｚｙｍｅ１２０Ｌ为重点，综合分析了外加酶糖化过程中影响麦汁总还原糖量和糖组成的各种因素，如各酶制剂的用量、糖化温度、料水比、物料比及各因素之间的相互作用等，确定了一套最优的糖化工艺方案。所得麦芽汁浸出率高、色度浅、粘度低、还原糖含量高；经过高效液相色谱分析（ＨＰＬＣ）表明，其糖组成合理；经过十天的发酵，发酵度达８２．２％，酒精分为６．４５５％（Ｗ／Ｗ）。     

The effects of three mash separation systems on the isomerisation of hop alpha-acids

This study investigates the effects of wort composition from three lautering systems on hop utilisation at different hop boiling and dosing times. A response surface methodology was applied with 60 single tests at a 5 litre scale. The parameters, which were varied, were lautering system, boiling time without hops, boiling time with hops and α-acid dosage. It was shown that the wort composition from the different lautering systems requires different boiling times or enables the reduction in boiling time with hops. Although the pH and original gravity of the lauter tun and mash filter worts were similar, different boiling times were necessary to achieve the same concentration of iso-α-acids. Further, there were variations in fatty acid composition of the worts. In order to be able to assess the effects on a larger scale, six brews were performed in a 10 hL pilot brewery. The utilisation of hop bitter substances differed despite the same boiling time and the same α-acid dosage in relation to the total quantity of wort. In addition, no significant losses of hop bitter substances were observed in the wort from a continuous mash filtration system due to the process related higher dosage of α-acid. Both sets of experiments showed that the boiling times depend on the wort composition and increased as follows: novel continuous mash filtration system < mash filter < lauter tun. The results lay the foundation for calculating the optimal parameter settings for each brewery to optimise the hop isomerisation rate. © 2020 The Authors. Journal of the Institute of Brewing published by John Wiley & Sons Ltd on behalf of The Institute of Brewing & Distilling     

探讨革新的糖化生产技术与设备

<正> 当今先进的技术已将啤酒酿造工艺带进了另一个新国度。今天,高质量的麦芽糖化工艺和低麦汁浊度已成为啤酒质量和味道稳定性的重要指标。 以融汇新技术知识为基础,针对于设备的功能性和有效利用性,ZIEMANN公司不断在改进其糖化设备     

Release of phenolic flavour precursors during wort production: Influence of process parameters and grist composition on ferulic acid release during brewing

In this study, the effects of mashing variables such as mashing-in temperature, time and pH, mash thickness, grist coarseness and composition, and stirring regime on the release of ferulic acid were examined. Ferulic acid is a precursor for the formation of flavour-active volatile phenols and a potent natural antioxidant in beer. Given one barley malt variety, the multitude of choice in setting various process parameters and adding brewery adjuncts during brewhouse operations can give rise to worts with widely varying ferulic acid levels. A clear difference in temperature- and pH-dependence between the release of the water-extracted and the enzymatically hydrolyzed fraction was found. The T,t-dependencies of arabinoxylan-degrading enzyme activities were correlated with ferulic acid release during mashing. Results from laboratory-scale mashing experiments were validated with those from a pilot-scale (5 h) wort production process. Enhancing the enzymatic release of phenolic flavour precursors from bound forms during mashing can greatly enhance the phenolic aroma potential of wort. Optimising this precursor release during mashing may be a means for controlling final volatile phenol levels in beer.     

Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten‐free Beverages and Beers

Proso millet is a gluten‐free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH‐adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The α‐amylase enzyme showed highest activity at a temperature of 60°C and at pH 5.0, whereas the β‐amylase activity was optimum at 40°C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50°C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68°C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control.     

Impact of buffering capacity on the acidification of wort by brewing‐relevant lactic acid bacteria

Acidified wort produced biologically using lactic acid bacteria (LAB) has application during sour beer production and in breweries adhering to the German purity law (Reinheitsgebot ). LAB cultures, however, suffer from end product inhibition and low pH, leading to inefficient lactic acid (LA) yields. Three brewing‐relevant LAB (Pediococcus acidilactici AB39, Lactobacillus amylovorus FST2.11 and Lactobacillus plantarum FST1.7) were examined during batch fermentation of wort possessing increasing buffering capacities (BC). Bacterial growth was progressively impaired when exposed to higher LA concentrations, ceasing in the pH range of 2.9–3.4. The proteolytic rest (50°C) during mashing was found to be a major factor improving the BC of wort. Both a longer mashing profile and the addition of an external protease increased the BC (1.21 and 1.24, respectively) compared with a control wort (1.18), and a positive, linear correlation (R ² = 0.957) between free amino nitrogen and BC was established. Higher levels of BC led to significant greater LA concentration (up to +24%) after 48 h of fermentation, reaching a maximal value of 11.3 g/L. Even higher LA (maximum 12.8 g/L) could be obtained when external buffers were added to wort, while depletion of micronutrient(s) (monosaccharides, amino acids and/or other unidentified compounds) was suggested as the cause of LAB growth cessation. Overall, a significant improvement in LA production during batch fermentation of wort is possible when BC is improved through mashing and/or inclusion of additives (protease and/or external buffers), with further potential for optimization when strain‐dependent nutritional requirements, e.g. sugar and amino acids, are considered. Copyright © 2017 The Institute of Brewing & Distilling     

The simultaneous saccharification and fermentation of malt dust and use in the acidification of mash

In brewing, the mash or wort is frequently acidified by the addition of lactic acid or the bioacidification of the mash. The present study provides an alternative approach for mash or wort acidification by the simultaneous saccharification and fermentation (SSF) of malt dust. In this method, fermentable carbohydrates released by the enzymatic breakdown of the cellulosic portion of the malt dust are converted to lactic acid by lactic acid bacteria. The effect of temperature, ranging between 45 and 51°C, solid loading of malt dust at 2, 5 and 10% (w/v) on a dry basis, and enzyme loading at 0.65, 2.6 and 6.5 filter paper units (FPU) per gram malt dust on SSF and change in pH in mash acidification were examined. The final pH and lactic acid concentration and final glucose concentration of the SSF media were significantly affected by the temperature of the process (p < 0.05). The highest lactic acid titre (9.7 g/L) and the lowest pH (3.12) were obtained by SSF of 10% (w/v) malt dust at 45°C with 6.5 FPU/g. The pH of the mashing solution [containing 20% (w/v) ground malt] decreased to around 5.4 and 5.2 after adding 1.9 and 2.9% of SSF media with pH 3.39. © 2019 The Institute of Brewing & Distilling